[0001] The present invention relates to a method for classifying the variety of malting
barley or malt using gene diagnosis and primers used for said method.
[0002] For variety classification of barley and malt, there has been conventionally used
a method for classifying the variety by comparing an SDS polyacrylamide gel electrophoretic
pattern of hordein and esterase contained therein. In addition, a classification method
using gene diagnosis has recently been developed (e.g., Chee et al., J. Am. Soc. Brew.
Chem., 51, 93 (1993)).
[0003] However, the variety classification method by way of comparing the electrophoretic
pattern of hordein and esterase is not necessarily to be an accurate classification
method, because the electrophoretic pattern may be modified according to growing conditions
of barley or due to the degradation of the hordein and esterase by protease during
malting process. Furthermore, since most of classification methods using gene diagnosis
use genes from unidentified origin as probe or primer, there has been a problem that
results obtained by the method cannot be directly correlated with the effect on the
quality of brew, even though mutation of materials or contamination of materials with
other varieties are indicated.
[0004] The present invention has been made considering the problem described above, and
aims to provice a more satisfactory method for classifying barley or malt using gene
diagnosis from the viewpoint of breeding melting barley or quality control of brewing
materials.
[0005] The solution to this problem is achieved by the provision of the embodiments characterised
by the patent claims.
[0006] In view of the situations described above, through continual ardent studies, the
present inventors identified sites wherein the base sequence differs among varieties
in genes which are important for brewing, and accomplished the present invention.
[0007] That is, the present invention provides a variety classification method for barley
or malt by performing polymerase chain reaction (PCR) with a set of primers designed
to flank the site of a gene which is important for brewing, wherein the base sequence
of the gene is made different among varieties so as to amplify the genomic DNA of
barley or malt, and classifying the variety of barley or malt based on the difference
in base sequence of the amplified DNA.
[0008] In a preferred embodiment of the present invention the gene important for brewing
which is used for amplification by PCR is a β-amylase gene, an α-amylase gene, a β-glucanase
gene or a gene encoding B1-Hordein. Oligonucleotides useful for the amplification
of these genes are depicted in Table 1. According to the invention it is possible
to use any primer set alone in order to amplify only one of the genes of interest
or to use combinations of the primer sets in order to amplify any possible combination
of two or more of the genes of interest. Furthermore, it is possible to use oligonucleotides
having sequences complementary to those depicted in Table 1.

[0009] The present invention also provides primers used for the variety classification method.
Primers according to the present invention can be synthesized with a commercial automated
DNA synthesizer using the β-cyanoethylphosphoamidide method or thioohosphite method.
[0010] More precisely, the present invention provides a variety classification method for
barley or malt comprising amplification of the genomic DNA of barley or malt by PCR
with the primer having the base sequence complementary to the gene which is important
gene in the brewing, and examination of the difference of base sequence of the amplified
DNA.
[0011] The present invention also provides a variety classification method for barley or
malt comprising the amplification of genomic DNA of barley or malt by PCR, which is
performed with either a set of oligonucleotides consisting of the sequence of (1)
5'-TTCAAAGCAGCAGCAGCG-3' (SEQ ID NO: 1) and (2) 5'-TTCTTCTGGTGCGCTCATC-3' (SEQ ID
NO: 2) or a set of oligonucleotides composed of the sequence complementary to the
nucleotides as the essential primer, and also a set of oligonucleotides consisting
of the sequence of (3) 5'-ATAAGTGGCCATCAATTCGGC-3' (SEQ ID NO: 3) and (4) 5'-GTGTGTCTGGCCAGGTAT-3'
(SEQ ID NO: 4) or a set of oligonucleotides composed of the sequence complementary
to them as the selective essential primer, and using either one of the two sets of
essential primers and either one of the two sets of selective essential primers or
either one primer thereof, and the classification based on the difference in the base
sequence of said DNA.
[0012] Furthermore, the present invention provides a variety classification method for barley
or malt comprising the amplification of the genomic DNA of barley or malt by PCR,
which is performed with oligonucleotides consisting of the sequence of (5) 5'-CGTGAAAAAACCGCCGCCGA-3'
(SEQ ID NO: 5), (6) 5'-CTTTCTCTCTCTAGCTGCGT-3' (SEQ ID NO: 6), (7) 5'-CCACCATGAAGACCTTCCTC-3'
(SEQ ID NO: 7) and (8) 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO: 8) or oligonucleotide
composed of the sequence complementary to them as a group of selective primers and
further using, in addition to the essential primers and the selective essential primers,
a combination of at least any one or any two primers from the group of selective primers,
of and the classification of a variety of barley or malt based on the difference in
the base sequence of the amplified DNA.
[0013] Furthermore, the present invention provides oligonucleotides useful as PCR primers
comprising nucleotide sequences as depicted in any one of the sequence listings SEQ
ID NO:1 to 8 or sequences complementary to these nucleotide sequences. The present
invention also relates to the use of the oligonucleotides according to the invention
as PCR primers.
[0014] According to the present invention, the genomic DNA is first extracted from the sample
of barley or malt. Extraction of the genomic DNA may be carried out, for example,
by a CTAB method (Nucleic Acids Res., 8, 4321 (1980)). Then, a portion of the targeted
gene is amplified by applying the primer of the present invention to the genomic DNA.
The partial amplification of the genomic DNA may be carried out, for example, by PCR
(Science, 230, 1350 (1985)). Then, the variety of barley or malt is classified either
by the base sequence determination of amplified DNA thus obtained or based on the
difference in the base sequence detected by electrophoresis on denatured gradient
gel or temperature gradient gel, or on the restriction enzyme cleavage pattern.
[0015] Since the method of the present invention aims to target the gene which is important
for brewing, it is highly possible that results obtained may directly influence the
quality of brew. Therefore, the method may become a satisfactory variety classification
method from the viewpoint of breeding of brewer's barley or the quality control of
brewing material.
[0016] Fig. 1 is a photograph of the polyacrylamide gel electrophoretic pattern of DNAs
which were amplified by PCR with primers (1) and (2), and then treated with restriction
enzymes NcoI and EcoT22I. In the figure, 1 to 10 correspond to the variety of barley,
A, B, B', C, and C' denote the type of electrophoretic pattern, and M is DNA MW marker
9 (Nippon Gene).
[0017] Fig. 2 is a photograph of the polyacrylamide gel electrophoretic pattern of DNAs
which were amplified by PCR with primers (3) and (4), and then treated with restriction
enzyme TaqI. In the figure, A, B and C denote the type of electrophoretic pattern
and M is DNA MW marker 9 (Nippon Gene).
[0018] Fig. 3 is a photograph of polyacrylamide gel electrophoretic pattern of DNAs which
were amplified by PCR with primers (5) and (6), and then treated with restriction
enzyme HaeIII. In the figure, A and B denote the type of electrophoretic pattern and
M is DNA MW marker 9 (Nippon Gene).
[0019] Fig. 4 is a photography of polyacrylamide gel electrophoretic pattern of DNAs which
are amplified by PCR with primers (7) and (8) (left half, A to E) and those which
were then treated with restriction enzyme HaeIII (right half, A to E). In the figure,
A, B, C, D and E denote the type of polyacrylamide gel electrophoretic pattern, and
M is DNA MW marker 9 (Nippon Gene) and M' DNA MW marker 2 (Nippon Gene).
[0020] The invention will now be described with reference to specific examples, however,
it should understood that the technical scope of the invention is not to be construed
as being limited to them in any way.
Example 1
Extraction of the Genomic DNA
[0021] In this embodiment, as the variety of barley or malt, Amagi Nijo (called Variety
No. 1 hereinafter), Haruna Nijo (called Variety No. 2 hereinafter), Misato Golden
(Variety No. 3 hereinafter), Clipper (called Variety No. 4 hereinafter), Schooner
(called Variety No. 5 hereinafter), Stirling (called Variety No. 6 hereinafter), Harrington
(called Variety No. 7 hereinafter), Manley (called Variety No. 8 hereinafter), Ellice
(called Variety No. 9 hereinafter) and Alexis (called Variety No. 10 hereinafter)
were used.
[0022] Embryos of barley or leaf buds of malt were taken out and the genomic DNA was extracted
from them using "Plant Genome Extraction Kit" (Clontech).
Example 2
Design and synthesis of primer
[0023] Various primers were designed from known base sequences of the barley genes important
for brewing, including those of β-amylase (J. Biochem., 115, 47 (1994)), α-amylase
(Plant Mol. Biol., 12, 119 (1989)), β-glucanase (Eur. J. Biochem., 194, 831 (1990)),
and B1-hordein (Nucleic Acid Res., 13, 7327 (1985)). PCR was performed with these
primers, and DNAs thus amplified were examined for the difference in the base sequence
among varieties using temperature gradient gel electrophoresis or based on the base
sequence determination.
[0024] As a result, it became clear that, using the primer (1) to (8), the DNA region wherein
the base sequence is different among varieties can be amplified, and utilizing the
restriction enzyme site in that region, the variety classification of barley or malt
may become possible. Synthesis and purification of primers were entrusted to Sawady
Technology Co. Ltd.
Example 3
Variety classification method using primer (1) and (2)
[0025] A PCR mixture (100 µl) which contained the genomic DNA (100 ng) extracted from 10
barley grains of each variety, dNTPs (20 nmol each), primers (1) and (2) (10 pmol
each) and Taq DNA polymerase (2.5 U) was subjected to 33 cycles of reaction wherein
each cycle consisted of incubating the mixture in sequence at 94°C for 1 min, 55°C
for 2 min, and 72°C for 1 min, and then finally treated at 72°C for 5 min. After the
completion of the PCR, restriction enzymes NcoI and EcoT22I (5 U each) and a buffer
for the enzymatic reaction were added to the reaction mixture (8 µl), and the mixture
was incubated at 37°C for 1 h. This reaction mixture was electrophoresed on 5% polyacrylamide
gel. After the electrophoresis, the gel was stained with ethidium bromide, and then
the DNA were made visible by UV exposure. Results are shown in Fig. 1. As shown in
this figure, from the electrophoretic pattern of the fragments of DNAs obtained by
digestion with restriction enzymes NcoI and EcoT22I, 10 varieties of barley could
be classified into 5 types (A, B, B', C and C'). Furthermore, based on results of
analyses on single grains, Variety Nos 5 and 10 were found to be a mixed type consisting
of either C and C' or B and C, therefore denoted as C/C' and B/C respectively.
Example 4
Variety classification method using primers (3) and (4)
[0026] Analysis was performed under similar conditions to those described for Example 3,
except that PCR was performed with the primer sequences (3) and (4) instead of (1)
and (2) and subjected to 30 cycles instead of 33, and the restriction enzyme digestion
was carried out with TaqI at 65°C instead of NcoI and EcoT22I at 37°C. As a result,
as shown in Fig. 2, the electrophoretic pattern could be classified into 3 types (A,
B and C).
Example 5
Variety classification method using primers (5) and (6)
[0027] Analysis was performed under similar conditions to the described for Example 3, except
that PCR was performed with the primer sequences (5) and (6) instead of (1) and (2)
and subjected to 30 cycles instead of 33, and restriction enzyme digestion was carried
out with HaeIII instead of NcoI and EcoT22I. As a result, as shown in Fig. 3, the
electrophoretic pattern could be classified into 2 types (A and B).
Example 6
Variety classification method using primers (7) and (8)
[0028] PCR was performed under similar conditions to those described for Example 3, except
for using the primer sequences (7) and (8), subjected to 30 cycles instead of 33,
and annealing at 57°C instead of 55°C. A portion of the PCR products were electrophresed
and the result was as shown on the left half of Fig. 4. Then, the PCR products were
digested with the restriction enzyme HaeIII under the similar conditions to those
described for Example 3, and electrophoresed. The fragment patterns were as shown
on the right half of Fig. 4. By comparing the results from intact and digested PCR
products, as shown in Fig. 4, the electrophoresis pattern could be classified into
5 types (A, B, C, D and E).
Example 7
Variety classification method by overall evaluation
[0029] Results of the type classification performed in Examples 3 to 6 are summarized in
Table 2. These results show that it is possible to classify all of the 10 variesties
by using the overall evaluation.

Example 8
Purity test of variety
[0030] Out of barley or malt purchased, 100 grains or 100 leaf buds as one sample lot were
subjected to analysis of type classification described above in Examples 3 to 6. As
a result, DNA fragments corresponding to the type of variety indicated at the time
of purchase were identified, and the purity of variety could be determined by examining
whether DNA fragments were contaminated with different type to those derived from
other varieties.
[0031] When contamination with other varieties is expected, the purity of the sample can
be estimated to a certain extent by quantifying the intensity of an electrophoretic
band with an image analyzer. Furthermore, when each single grain or leaf bud is subjected
to similar analysis, the extent of contamination and type of contaminating variety
may be possibly determined qualitatively as well as quantitatively.
1. A method for classifying a variety of barley or malt by amplifying genomic DNA of
barley or malt by PCR with one or more pairs of primers comprising nucleotide sequences
complementary to one or more genes important for brewing, and examining differences
in the nucleotide sequence of the amplified DNA.
2. The method of claim 1, wherein the gene(s) that is (are) important for brewing is
(are) a β-amylase gene, an α-amylase gene, a β-glucanase gene, a gene coding for B1-hordein
or a combination of these genes.
3. The method of claim 1 or 2, wherein the amplification is performed with a pair of
oligonucleotides as primers which comprise the nucleotide sequence
(1) 5'-TTCAAAGCAGCAGCAGCG-3 (SEQ ID NO:1) and
(2) 5'-TTCTTCTGGTGCGCTCATC-3' (SEQ ID NO:2),
respectively, or nucleotide sequences complementary to these sequences.
4. The method of any one of claims 1 to 3, wherein the amplification is performed with
a pair of oligonucleotides as primers which comprise the nucleotide sequence
(3) 5'-ATAAGTGGGCATCAATTCGGC-3' (SEQ ID NO:3) and
(4) 5'-GTGTGTCTGGCCAGGTAT-3' (SEQ ID NO:4),
respectively, or nucleotide sequences complementary to these sequences.
5. The method of any one of claims 1 to 4, wherein amplification is performed with a
pair of oligonucleotides as primers which comprise the nucleotide sequence
(5) 5'-CGTGAAAAAACCGCCGCCGA-3' (SEQ ID NO:5) and
(6) 5'-CTTTCTCTCTCTAGCTGCGT-3' (SEQ ID NO:6),
respectively, or nucleotide sequences complementary to these sequences.
6. The method of any one of claims 1 to 5, wherein amplification is performed with a
pair of oligonucleotides as primers which comprise the nucleotide sequence
(7) 5'-CCACCATGAAGACCTTCCTC-3' (SEQ ID NO:7), and
(8) 5'-TCGCAGGATCCTGTACAACG-3' (SEQ ID NO:8),
respectively, or nucleotide sequences complementary to these sequences.
7. An oligonucleotide comprising a nucleotide sequence as depicted in any one of the
sequence listings SEQ NO:1 to 6 or a nucleotide sequence complementary to any one
of these sequences.
8. Use of an oligonucleotide of claim 7 as a PCR primer.